Polynucleotides encoding aminolevulinic acid biosynthetic enzymes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding an aminolevulinic acid biosynthetic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the aminolevulinic acid biosynthetic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the aminolevulinic acid biosynthetic enzyme in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/146,600, filed Jul. 30, 1999.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingaminolevulinic acid biosynthetic enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

A major regulatory point in the biosynthesis of tetrapyrrolic pigmentslike chlorophyll and heme is the formation of the building block5-aminolevulinic acid (ALA) which provides all the carbon and nitrogenatoms of the tetrapyrrole ring. There are two different routes by whichALA is synthesized in the living cell. In animals, fungi and someeubacteria, succinyl-CoA and glycine are condensed by ALA synthase toyield ALA with the concomitant liberation of the carboxyl carbon ofglycine as carbon dioxide. In contrast, in plants, algae and certaineubacteria, ALA is formed from Glu-tRNA^(Glu) via two enzymaticreactions (Jahn et al. (1992) Trends Biochem Sci 17:215-218). First,Glu-tRNA reductase converts Glu-tRNA^(Glu) to glutamate 1-semialdehyde(GSA) with the concomitant release of tRNA^(Glu). GSA aminotransferasethen converts GSA to ALA.

Given the facts that plants and animals differ in the way theysynthesize ALA and that ALA is an essential compound for survival, it isenvisioned that inhibitors of Glu-tRNA reductase and GSAaminotransferase may serve as effective herbicides that are nontoxic toman and other animals. Genes encoding Glu-tRNA reductase and GSAaminotransferase may be isolated and then overexpressed in bacterial oryeast hosts to provide the huge amounts of protein that is needed forinhibitor discovery and design.

SUMMARY OF THE INVENTION

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 25 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide of SEQ ID NO:18; (b) a second nucleotidesequence encoding a polypeptide of at least 25 amino acids having atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:10; (c) a third nucleotidesequence encoding a polypeptide of at least 40 amino acids having atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:14; (d) a fourth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:28; (e) a fifth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 90% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:24; (f) a sixth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:2; (g) a seventh nucleotidesequence encoding a polypeptide of at least 80 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:6; (h) an eighth nucleotidesequence encoding a polypeptide of at least 240 amino acids having atleast 90% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:22; (i) a ninth nucleotidesequence encoding a polypeptide of at least 250 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:16; (j) a tenth nucleotidesequence encoding a polypeptide of at least 300 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:4 and 26; (k) an eleventh nucleotide sequence encoding a polypeptideof at least 500 amino acids having at least 90% identity based on theClustal method of alignment when compared to a polypeptide of SEQ IDNO:12; and (1) a twelfth nucleotide sequence comprising the complementof (a), (b), (c), (d), (e), (f), (g), (h), (i), G) or (k).

In a second embodiment, it is preferred that the isolated polynucleotideof the claimed invention comprises a first nucleotide sequence whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:1, 3, 5, 9, 11, 13, 15, 17, 21, 23, 25, and 27 that codes forthe polypeptide selected from the group consisting of SEQ ID NOs:2, 4,6, 10, 12, 14, 16, 18, 22, 24, 26, and 28.

In a third embodiment, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence of at least one of 60(preferably at least one of 40, most preferably at least one of 30)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 9, 11, 13, 15, 17, 21, 23,25, and 27 and the complement of such nucleotide sequences.

In a fourth embodiment, this invention relates to a chimeric genecomprising an isolated polynucleotide of the present invention operablylinked to at least one suitable regulatory sequence.

In a fifth embodiment, the present invention concerns an isolated hostcell comprising a chimeric gene of the present invention or an isolatedpolynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

In a sixth embodiment, the invention also relates to a process forproducing an isolated host cell comprising a chimeric gene of thepresent invention or an isolated polynucleotide of the presentinvention, the process comprising either transforming or transfecting anisolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the present invention.

In a seventh embodiment, the invention concerns a Glu-tRNA reductase orGSA aminotransferase polypeptide selected from the group consisting of:(a) a polypeptide of at least 25 amino acids having at least 80%identity based on the Clustal method of alignment when compared to apolypeptide of SEQ ID NO:18; (b) a polypeptide of at least 25 aminoacids having at least 85% identity based on the Clustal method ofalignment when compared to a polypeptide of SEQ ID NO:10; (c) apolypeptide of at least 40 amino acids having at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:14; (d) a polypeptide of at least 50 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:28; (e) a polypeptide of at least50 amino acids having at least 90% identity is based on the Clustalmethod of alignment when compared to a polypeptide of SEQ ID NO:24; (f)a polypeptide of at least 50 amino acids having at least 95% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:2; (g) a polypeptide of at least 80 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:6; (h) a polypeptide of at least240 amino acids having at least 900/c identity based on the Clustalmethod of alignment when compared to a polypeptide of SEQ ID NO:22; (i)a polypeptide of at least 250 amino acids having at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:16; (j) a polypeptide of at least 300 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:4 and 26; and (k) a polypeptide of at least 500 amino acids havingat least 90% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:12.

In an eighth embodiment, the invention relates to a method of selectingan isolated polynucleotide that affects the level of expression of aGlu-tRNA reductase or a GSA aminotransferase polypeptide or enzymeactivity in a host cell, preferably a plant cell, the method comprisingthe steps of: (a) constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention; (b)introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; (c) measuring the level of the Glu-tRNA reductase or aGSA aminotransferase polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and (d) comparing the level ofthe Glu-tRNA reductase or a GSA aminotransferase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide withthe level of the Glu-tRNA reductase or a GSA aminotransferasepolypeptide or enzyme activity in the host cell that does not containthe isolated polynucleotide.

In a ninth embodiment, the invention concerns a method of obtaining anucleic acid fragment encoding a substantial portion of a Glu-tRNAreductase or a GSA aminotransferase polypeptide, preferably a plantGlu-tRNA reductase or a GSA aminotransferase polypeptide, comprising thesteps of: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 9, 11, 13, 15, 17, 21, 23, 25, and 27 and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a substantialportion of a Glu-tRNA reductase or a GSA aminotransferase amino acidsequence.

In a tenth embodiment, this invention relates to a method of obtaining anucleic acid fragment encoding all or a substantial portion of the aminoacid sequence encoding a Glu-tRNA reductase or a GSA aminotransferasepolypeptide comprising the steps of: probing a cDNA or genomic librarywith an isolated polynucleotide of the present invention; iv identifyinga DNA clone that hybridizes with an isolated polynucleotide of thepresent invention; isolating the identified DNA clone; and sequencingthe cDNA or genomic fragment that comprises the isolated DNA clone.

In an eleventh embodiment, this invention concerns a composition, suchas a hybridization mixture, comprising an isolated polynucleotide of thepresent invention.

In a twelfth embodiment, this invention concerns a method for positiveselection of a transformed cell comprising: (a) transforming a host cellwith the chimeric gene of the present invention or an expressioncassette of the present invention; and (b) growing the transformed hostcell, preferably a plant cell, such as a monocot or a dicot, underconditions which allow expression of the Glu-tRNA reductase or the GSAaminotransferase polynucleotide in an amount sufficient to complement anull mutant to provide a positive selection means.

In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of an aminolevulinic acid biosyntheticenzyme in a host cell comprising: (a) transforming a host cell with achimeric gene of the present invention; and (b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of the aminolevulinic acid biosyntheticenzyme in the transformed host cell.

A further embodiment of the instant invention is a method for evaluatingat least one compound for its ability to inhibit the activity of anaminolevulinic acid biosynthetic enzyme, the method comprising the stepsof: (a) transforming a host cell with a chimeric gene comprising anucleic acid fragment encoding an aminolevulinic acid biosyntheticenzyme polypeptide, operably linked to suitable regulatory sequences;(b) growing the transformed host cell under conditions that are suitablefor expression of the chimeric gene wherein expression of the chimericgene results in production of an aminolevulinic acid biosynthetic enzymein the transformed host cell; (c) optionally purifying theaminolevulinic acid biosynthetic enzyme polypeptide expressed by thetransformed host cell; (d) treating the aminolevulinic acid biosyntheticenzyme polypeptide with a compound to be tested; and (e) comparing theactivity of the aminolevulinic acid biosynthetic enzyme polypeptide thathas been treated with a test compound to the activity of an untreatedaminolevulinic acid biosynthetic enzyme polypeptide, thereby selectingcompounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A-1B present an alignment of amino acid sequences of Glu-tRNAreductase encoded by the nucleotide sequences derived from corn clonecsc1c.pk005.i15 (SEQ ID NO:4) and soybean clone sfl1.pk0060.c4 (SEQ IDNO:12), and the Glu-tRNA reductase from Glycine max (NCBI GI No.4324495; SEQ ID NO:29). Amino acids which are conserved among all and atleast two sequences with an amino acid at that position are indicatedwith an asterisk (*). Dashes are used by the program to maximizealignment of the sequences.

FIGS. 2A-2B present an alignment of amino acid sequences of GSAaminotransferase encoded by the nucleotide sequence derived from riceclone rl0n.pk0078.b9 (SEQ ID NO:26) and the GSA aminotransferase fromHordeum vulgare (NCBI GI No. 1170029; SEQ ID NO:30). Amino acids whichare conserved between the two sequences are indicated with an asterisk(*). Dashes are used by the program to maximize alignment of thesequences.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig*”), contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding the entire protein derived froman FIS, a contig, or an FIS and PCR (“CGS”). Nucleotide SEQ ID NOs:1, 5,9, 13, 17, 23, and 27 correspond to nucleotide SEQ ID NOs:1, 3, 5, 7, 9,13, and 17, respectively, presented in U.S. Provisional Application No.60/146,600, filed Jul. 30, 1999. Amino acid SEQ ID NOs:2, 6, 10, 14, 18,24, and 28 correspond to amino acid SEQ ID NOs:2, 4, 6, 8, 10, 14, and18, respectively, presented in U.S. Provisional Application No.60/146,600, filed Jul. 30, 1999. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R §1.821-1.825.

TABLE 1 Aminolevulinic Acid Biosynthetic Enzymes SEQ ID NO: Clone(Nucleo- (Amino Protein (Plant Source) Designation Status tide) Acid)Glu-tRNA Reductase p0008.cb3lk05r EST  1  2 (Corn) Glu-tRNA Reductasecsclc.pk005.i15 CGS  3  4 (Corn) (FIS) Glu-tRNA Reductaserlr48.pk0037.f4 EST  5  6 (Rice) Glu-tRNA Reductase rlr48.pk0037.f4 FIS 7  8 (Rice) Glu-tRNA Reductase sfl1.pk0060.c4 EST  9 10 (Soybean)Glu-tRNA Reductase sfl1.pk0060.c4 CGS 11 12 (Soybean) (FIS) Glu-tRNAReductase srr1c.pk001.p10 EST 13 14 (Soybean) Glu-tRNA Reductasesrr1c.pk001.p10 FIS 15 16 (Soybean) Glu-tRNA Reductase ses8w.pk0017.c6EST 17 18 (Soybean) Glu-tRNA Reductase wlln.pk0060.b11 FIS 19 20 (Wheat)GSA Aminotransferase cr1.pk0013.e7 FIS 21 22 (Corn) GSA Aminotransferaser10n.pk0078.b9 EST 23 24 (Rice) GSA Aminotransferase r10n.pk0078.b9 CGS25 26 (Rice) (FIS) GSA Aminotransferase wlm12.pk0015.d7 EST 27 28(Wheat)

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs:1, 3, 5, 9, 11, 13, 15, 17, 21, 23, 25, and 27,or the complement of such sequences.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as and notlimited to other chromosomal and extrachromosomal DNA and RNA. Isolatedpolynucleotides may be purified from a host cell in which they naturallyoccur. Conventional nucleic acid purification methods known to skilledartisans may be used to obtain isolated polynucleotides. The term alsoembraces recombinant polynucleotides and chemically synthesizedpolynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterns “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 9, 11, 13, 15, 17, 21, 23, 25, and 27 and the complement ofsuch nucleotide sequences may be used in methods of selecting anisolated polynucleotide that affects the expression of a Glu-tRNAreductase or a GSA aminotransferase polypeptide in a host cell. A methodof selecting an isolated polynucleotide that affects the level ofexpression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial) may comprise thesteps of: constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide with the level of a polypeptideor enzyme activity in a host cell that does not contain the isolatedpolynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min. and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1X SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 25, 40, or 50 amino acids, preferably atleast 80 or 100 amino acids, more preferably at least 150 amino acids,still more preferably at least 200 amino acids, and most preferably atleast 240, 250, 300, or 500 amino acids. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403410). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide, Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to con the entire desired nucleic acid fragment“Chemically synthesized”, as related to a nucleic acid fragment, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of nucleic acid fragments may be accomplished using wellestablished procedures, or automated chemical synthesis can be performedusing one of a number of commercially available machines. Accordingly,the nucleic acid fragments can be tailored for optimal gene expressionbased on optimization of the nucleotide sequence to reflect the codonbias of the host cell. The skilled artisan appreciates the likelihood ofsuccessful gene expression if codon usage is biased towards those codonsfavored by the host. Determination of preferred codons can be based on asurvey of genes derived from the host cell where sequence information isavailable.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ noncoding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ noncoding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Kienowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ noncoding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell which either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth Enzymol. 143:277) and particle-acceleratedor “gene gun” transformation technology (Klein et al. (1987) Nature(London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein byreference). Thus, isolated polynucleotides of the present invention canbe incorporated into recombinant constructs, typically DNA constructs,capable of introduction into and replication in a host cell. Such aconstruct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1989;and Flevin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990. Typically, plant expression vectors include, forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 25 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide of SEQ ID NO:18; (b) a second nucleotidesequence encoding a polypeptide of at least 25 amino acids having atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:10; (c) a third nucleotidesequence encoding a polypeptide of at least 40 amino acids having atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:14; (d) a fourth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 80% a identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:28; (e) a fifth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 90% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:24; (f) a sixth nucleotidesequence encoding a polypeptide of at least 50 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:2; (g) a seventh nucleotidesequence encoding a polypeptide of at least 80 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:6; (h) an eighth nucleotidesequence encoding a polypeptide of at least 240 amino acids having atleast 90% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:22; (i) a ninth nucleotidesequence encoding a polypeptide of at least 250 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:16; (j) a tenth nucleotidesequence encoding a polypeptide of at least 300 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:4 and 26; (k) an eleventh nucleotide sequence encoding a polypeptideof at least 500 amino acids having at least 90% identity based on theClustal method of alignment when compared to a polypeptide of SEQ IDNO:12; and (l) a twelfth nucleotide sequence comprising the complementof (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k).

Preferably, the first nucleotide sequence comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 9,11, 13, 15, 17, 21, 23, 25, and 27, that codes for the polypeptideselected from the group consisting of SEQ ID NOs:2, 4, 6, 10, 12, 14,16, 18, 22, 24, 26, and 28.

Nucleic acid fragments encoding at least a portion of severalaminolevulinic acid biosynthetic enzymes have been isolated andidentified by comparison of random plant cDNA sequences to publicdatabases containing nucleotide and protein sequences using the BLASTalgorithms well known to those skilled in the art. The nucleic acidfragments of the instant invention may be used to isolate cDNAs andgenes encoding homologous proteins from the same or other plant species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other Glu-tRNA reductase or a GSAaminotransferase, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, an entire sequence can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, end-labeling techniques, or RNA probes usingavailable in vitro transcription systems. In addition, specific primerscan be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate fill-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 9, 11, 13, 15,17, 21, 23, 25, and 27 and the complement of such nucleotide sequencesmay be used in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a Glu-tRNA reductase or a GSAaminotransferase polypeptide, preferably a substantial portion of aplant Glu-tRNA reductase or a GSA aminotransferase polypeptide,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 60 (preferably atleast one of 40, most preferably at least one of 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 9, 11, 13, 15, 17, 21, 23, 25, and 27,and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a portion of a Glu-tRNA reductase or a GSAaminotransferase polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesis. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of tetrapyrrolicpigments like heme and chlorophyll in those cells. The nucleic acidfragments of the instant invention may also be used for overexpressionin bacterial or yeast hosts, thereby efficiently producing large amountsof the encoded polypeptides which could then be used for screeningdifferent compounds for potential herbicidal activity.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Noncoding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintercellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the cosuppression or antisense chimeric genescould be introduced into plants via transformation wherein expression ofthe corresponding endogenous genes are reduced or eliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adormant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns a polypeptideselected from the group consisting of: (a) a polypeptide of at least 25amino acids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide of SEQ ID NO:18; (b) apolypeptide of at least 25 amino acids having at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:10; (c) a polypeptide of at least 40 amino acids having atleast 85% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:14; (d) a polypeptide of at least50 amino acids having at least 80% identity based on the Clustal methodof alignment when compared to a polypeptide of SEQ ID NO:28; (e) apolypeptide of at least 50 amino acids having at least 90% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:24; (f) a polypeptide of at least 50 amino acids having atleast 95% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:2; (g) a polypeptide of at least80 amino acids having at least 95% identity based on the Clustal methodof alignment when compared to a polypeptide of SEQ ID NO:6; (h) apolypeptide of at least 240 amino acids having at least 90% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO:22; (i) a polypeptide of at least 250 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide of SEQ ID NO:16; (j) a polypeptide of at least300 amino acids having at least 95% identity based on the Clustal methodof alignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:4 and 26; and k a polypeptide of at least 500amino acids having at least 90% identity based on the Clustal method ofalignment when compared to a polypeptide of SEQ ID NO:12.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded aminolevulinic acid biosynthetic enzyme. An example of avector for high level expression of the instant polypeptides in abacterial host is provided (Example 7).

Additionally, the instant polypeptides can be used as a target tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in aminolevulinicacid biosynthesis. Accordingly, inhibition of the activity of one ormore of the enzymes described herein could lead to inhibition of plantgrowth. Thus, the instant polypeptides could be appropriate for newherbicide discovery and design.

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hun. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Karazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:679-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean, andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat LibraryTissue Clone cr1 Corn Root From 7 Day Old Seedlings cr1.pk0013.e7 csc1cCorn 20 Day Old Seedling (Germination csc1c.pk005.i15 Cold Stress) p0008Corn 3 Week Old Leaf p0008.cb3lk05r rl0n Rice 15 Day Old Leaf*r10n.pk0078.b9 rlr48 Resistant Rice Leaf 15 Days After rlr48.pk0037.f4Germination, 48 Hours After Infection of Strain Magaporthe grisea4360-R-62 (AVR2-YAMO) ses8w Soybean Mature Embryo 8 Weeks Afterses8w.pk0017.c6 Subculture sfl1 Soybean Immature Flower sfl1.pk0060.c4srr1c Soybean 8 Day Old Root srr1c.pk001.p10 wl1n Wheat Leaf From 7 DayOld Seedling Light wl1n.pk0060.b11 Grown* wlm12 Wheat Seedling 12 HoursAfter Inoculation wlm12.pk0015.d7 With Erysiphe graminis f. sp tritici*These libraries were normalized essentially as described in U.S. Pat.No. 5,482,845, incorporated herein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding aminolevulinic acid biosynthetic enzyme wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The cDNA sequences obtained in Example 1 were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Glu-tRNA Reductase

The BLASTX search using the EST sequences from clones p0008.cb31k05r,rlr48.pk0037.f4, sfl1.pk0060.c4, srr1c.pk001.p10, and ses8w.pk0017.c6revealed similarity of the proteins encoded by the cDNAs to Glu-tRNAreductase from different plant species. The BLAST results for each ofthese ESTs are shown in Table 3:

TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous toGlu-tRNA Reductase BLAST Results NCBI GenBank pLog Clone OrganismIdentifier No. Score p0008.cb3lk05r Oryza sativa 2920320 23.7rlr48.pk0037.f4 Oryza sativa 2920320 44.0 sfl1.pk0060.c4 Glycine max4324495 24.2 srr1c.pk001.p10 Arabidopsis thaliana 1170203 13.0ses8w.pk0017.c6 Glycine max 4324495 11.5

The sequence of a portion of the cDNA insert from clone p0008.cb31k05ris shown in SEQ ID NO:1; the deduced amino acid sequence of this portionof the cDNA is shown in SEQ ID NO:2. The sequence of a portion of thecDNA insert from clone rlr48.pk0037.f4 is shown in SEQ ID NO:5; thededuced amino acid sequence of this portion of the cDNA is shown in SEQID NO:6. The sequence of a portion of the cDNA insert from clonesfl1.pk0060.c4 is shown in SEQ ID NO:9; the deduced amino acid sequenceof this portion of the cDNA is shown in SEQ ID NO:10. The sequence of aportion of the cDNA insert from clone srr1c.pk001.p10 is shown in SEQ IDNO:13; the deduced amino acid sequence of this portion of the cDNA isshown in SEQ ID NO:14. The sequence of a portion of the cDNA insert fromclone ses8w.pk0017.c6 is shown in SEQ ID NO:17; the deduced amino acidsequence of this portion of the cDNA is shown in SEQ ID NO:18. BLASTscores and probabilities indicate that the instant nucleic acidfragments encode portions of Glu-tRNA reductase. The sequence derivedfrom clone p0008.cb31k05r represents the first corn sequence encodingGlu-tRNA reductase. Nucleic acids encoding Glu-tRNA reductase have beenpreviously characterized in rice (Nakayashiki, T. and Inokuchi H.(1998), Plant Physiol. 117:332) and soybean (Sangwan I. and O'Brian, M.R. (1999), Plant Physiol. 119.593-598). Among the sequences disclosedherein, the rice Glu-tRNA reductase amino acid sequence reported inNakayashiki T. and Inokuchi H. (1998), Plant Physiol. 117:332 shows themost homology with SEQ ID NO:6, with 93.1% identity over a sequence of87 amino acids.

The sequence of the entire cDNA insert in some of the clones listed inTable 3 was determined. The BLASTX search using the EST sequences fromclones listed in Table 4 revealed similarity of the polypeptides encodedby the cDNAs to Glu-tRNA reductase from Oryza sativa (NCBI GenBankIdentifier (GI) No. 3913811), Cucumis sativus (NCBI GI No. 1346261),Hordeum vulgare (NCBI GI No. 1039332), and Glycine max (NCBI GI No.4324495). Shown in Table 4 are the BLAST results for individual ESTsC(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), the sequences of contigs assembled fromtwo or more ESTs (“Contig”), sequences of contigs assembled from an FISand one or more ESTs (“Contig*”), or sequences encoding an entireprotein derived from an FIS, a contig, or an FIS and PCR (“CGS”):

TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous toGlu-tRNA Reductase BLAST Results Clone Status NCBI GI No. pLog Scorecsc1c.pk005.i15 CGS 3913811 >254.00 (FIS) rlr48.pk0037.f4 FIS3913811 >254.00 sfl1.pk0060.c4 CGS 4324495 >254.00 (FIS) srr1c.pk001.p10FIS 1346261 >254.00 wl1n.pk0060.b11 FIS 1039332 >254.00

FIGS. 1A-1B present an alignment of the amino acid sequences set forthin SEQ ID NOs:4 and 12 and the Glycine max sequence (NCBI GI No.4324495; SEQ ID NO:29). The data in Table 5 represents a calculation ofthe percent identity of the amino acid sequences set forth in SEQ IDNOs:4 and 12 and the Glycine max sequence (NCBI GI No. 4324495; SEQ IDNO:29).

TABLE 5 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toGlu-tRNA Reductase Percent Identity to SEQ ID NO. NCBI GI No. 4324495;SEQ ID NO:29  4 66.5 12 84.3

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE, bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY-10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE I, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode all or a substantial portion of a Glu-tRNA reductase. Thesesequences represent the first corn and wheat sequences indicated toencode Glu-tRNA reductase known to Applicant.

Example 4 Characterization of cDNA Clones Encoding GSA Aminotransferase

The BLASTX search using the EST sequences from clones r10n.pk0078.b9 andwlm12.pk0015.d7 revealed similarity of the proteins encoded by the cDNAsto GSA aminotransferase from Hordeum vulgare (NCBI GI No. 11170029). TheBLAST results for each of these ESTs are shown in Table 6:

TABLE 6 BLAST Results for Clones Encoding Polypeptides Homologous to GSAAminotransferase BLAST Results Clone Organism NCBI GI No. pLog Scorer10n.pk0078.b9 Hordeum vulgare 1170029 34.2 wlm12.pk0015.d7 Hordeumvulgare 1170029 21.0

The sequence of a portion of the cDNA insert from clone r10 n.pk0078.b9is shown in SEQ ID NO:23; the deduced amino acid sequence of thisportion of the cDNA is shown in SEQ ID NO:24. The sequence of a portionof the cDNA insert from clone wlm12.pk0015.d7 is shown in SEQ ID NO:27;the deduced amino acid sequence of this portion of the cDNA is shown inSEQ ID NO:28. BLAST scores and probabilities indicate that the instantnucleic acid fragments encode portions of GSA aminotransferase. Thesesequences represent the first rice and wheat sequences encoding GSAaminotransferase known to Applicant. A nucleic acid fragment encodingGSA aminotransferase has been previously characterized in soybean(Sangwan, I. and O'Brian, M. R. (1993), Plant Physiol. 102:829-834), andthe amino acid sequence encoded by said nucleic acid fragment shows themost homology, among the sequences disclosed herein, to SEQ ID NO:16,with 93.8% identity over a sequence of 97 amino acids.

The sequence of the entire cDNA insert in clone r10n.pk0078.b9 listed inTable 6 was determined. Further sequencing and searching of the DuPontproprietary database allowed the identification of a corn clone encodingGSA aminotransferase. The BLASTX search using the EST sequences fromclones listed in Table 7 revealed similarity of the polypeptides encodedby the cDNAs to GSA aminotransferase from Hordeum vulgare (NCBI GI No.1170029). Shown in Table 7 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), the sequences of contigs assembled fromtwo or more ESTs (“Contig”), sequences of contigs assembled from an FISand one or more ESTs (“Contig*”), or sequences encoding an entireprotein derived from an FIS, a contig, or an FIS and PCR (“CGS”):

TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous toGSA Aminotransferase BLAST pLog Score Clone Status NCBI GI No. 1170029cr1.pk0013.e7 FIS 120.00 r10n.pk0078.b9 CGS >254.00 (FIS)

FIGS. 2A-2B present an alignment of the amino acid sequence set forth inSEQ ID NO:26 and the Hordeum vulgare sequence (NCBI GI No. 1170029; SEQID NO:30). The data in Table 8 represents a calculation of the percentidentity of the amino acid sequence set forth in SEQ ID NO:26 and theHordeum vulgare sequence (NCBI GI No. 1170029; SEQ ID NO:30).

TABLE 8 Percent identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toGSA Aminotransferase Percent Identity to SEQ ID NO. NCBI GI NO. 1170029;SEQ ID NO:30 26 89.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode all or a substantial portion of a GSA aminotransferase. Thesesequences represent the first corn, rice, and wheat sequences encodingGSA aminotransferase known to Applicant.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR The amplified DNA is then digested with restriction enzymesNcoI and SmaI and fractionated on an agarose gel. The appropriate bandcan be isolated from the gel and combined with a 4.9 kb NcoI-SmaIfragment of the plasmid pML103. Plasmid pML103 has been deposited underthe terms of the Budapest Treaty at ATCC (American Type CultureCollection, 10801 University Blvd., Manassas, Va. 20110-2209), and bearsaccession number ATCC 97366. The DNA segment from pML103 contains a 1.05kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene inthe vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at15° C. overnight, essentially as described (Maniatis). The ligated DNAmay then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptide, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)nay be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the 0 subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem 261:9228-9238) can be used for expression ofthe instant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translationinitiation codon and about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites Nco I (which includes the ATGtranslation initiation codon), Sma I, Kpn I and Xba I. The entirecassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 nm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS 1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight

Example 8 Evaluating Compounds for Their Ability to Inhibit the Activityof an Aminolevulinic Acid Biosynthetic Enzyme

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 7, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfite precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by lining the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fission protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for Glu-tRNA reductase are presented byJahn, D. et al. (1991), J. Biol. Chem. 266:2542-2548. Assays for GSAaminotransferase are presented by Jahn, D. et al. (1991), J. Biol. Chem266:161-167.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding polypeptide with Glu-tRNA Reductase activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal method of alignment, when compared to oneof SEQ ID NO:4, or (b) a complement of the nucleotide sequence, whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.
 2. The polynucleotide of claim1, wherein the amino acid sequence of the polypeptide comprises SEQ IDNO:4.
 3. The polynucleotide of claim 1 wherein the nucleotide sequencecomprises SEQ ID NO:3.
 4. A vector comprising the polynucleotide ofclaim
 1. 5. A recombinant DNA construct comprising the polynucleotide ofclaim 1 operably linked to at least one regulatory sequence.
 6. A methodfor transforming a cell, comprising transforming a cell with thepolynucleotide of claim
 1. 7. A cell comprising the recombinant DNAconstruct of claim
 5. 8. A method for producing a plant comprisingtransforming a plant cell with the polynucleotide of claim 1 andregenerating a plant from the transformed plant cell.
 9. A plantcomprising the recombinant DNA construct of claim
 5. 10. A seedcomprising the recombinant DNA construct of claim 5.